Enhancement of Blood Stasis and Vascular Permeability in Meth-A Tumors by Administration of Hyperthermia in Combination with Tumor Necrosis Factor

Blood stasis and vascular permeability induced by tumor necrosis factor (TNF) in Meth-A tumors transplanted in BALB/c mice were significantly enhanced by hyperthermia at 40°C for 30 min immediately following TNF administration. A dose-dependent, sustained decline in the intratumoral blood flow rate occurred following the administration of TNF alone (i.v.; 5 × 10³, 5 × 10⁴, or 5 × 10⁵ JRU/kg) and was enhanced by the administration of hyperthermia in combination with the TNF, even though no decline occurred with hyperthermia alone. The combination of TNF at 5 × 10⁵ JRU/kg and hyperthermia resulted in a blood flow ratio (ratio of blood flow after administration to that before) of 0.47 at 1 h compared with a ratio of 0.65 at 1 h after TNF alone. The blood flow in normal skin sites did not decrease in any case. The permeability of the intratumoral vasculature similarly increased in a dose-dependent manner after the administration of TNF alone and was further increased by combination with hyperthermia, even though no increase occurred with hyperthermia alone. The mean permeability in mice receiving TNF alone at 5 × 10⁵ JRU/kg was 1.35 times that in untreated mice. In mice receiving TNF at the same dose together with hyperthermia, the ratio was increased to 1.65. The results suggest that TNF selectively suppresses intratumoral blood flow, that this effect is enhanced by mild hyperthermia, and that the mechanism of the suppression by TNF with or without hyperthermia partly involves an increase in blood vessel permeability.     

Enhancement of Lysosomal Enzyme Activity by Recombinant Human Tumor Necrosis Factor and Its Role in Tumor Cell Killing in vitro

We investigated the effect of recombinant human tumor necrosis factor (TNF) on the lysosomal enzyme activity of various established cell lines in vitro. Incubation of 1 × 10⁶TNF-sensitive mouse tumorigenic fibroblasts (L-M cells) in the presence of TNF (100 U/ml) for 48 h increased the total (the sum of the enzyme activities in the lysosomes and the cytoplasm) acid phosphatase and /9-ghicuronidase activities by 3.7- and 4.2-fold, respectively. The same increase was observed even when 1 U/ml of TNF was added to some cultures and no further augmentation occurred at 10 or 100 U/ml. Measurement of total and free enzyme activities showed that TNF stimulation not only enhanced the total intracellular enzyme activity but also accelerated the conversion into free (cytoplasmic) enzyme activity. Addition of a lysosomotropic agent (methylamine) suppressed both the enhancement of lysosomal enzyme activity and the cytotoxicity of TNF. A similar enhancement of lysosomal enzyme activities was also detected in various TNF-sensitive tumor cell lines, and a strong correlation (acid phosphatase: r= 0.836, β-glucuronidase: r=0.910) was observed between the enhancement of enzyme activity and sensitivity to TNF. No such increase was detected in TNF-resistant human diploid cells. These results show that TNF induces the activation and release of lysosomal enzymes in TNF-sensitive cells, and suggest that such events may play an important role in TNF-mediated cytotoxicity.     

Enhancement of Cytotoxicity of Cisplatin in vitro by Recombinant Human Tumor Necrosis Factor and/or Recombinant Human Interferon-α, β and -γ

This study was conducted to investigate the modulatory effects of recombinant human tumor necrosis factor (rH-TNF) and recombinant human interferon (rH-IFN)-α, -β and -γ, either alone or in combination, on the cytotoxicity of cisplatin, using MTT assay, against MKN-45 (human stomach adenocarcinoma). MKN-45 was resistant to rH-TNF even at doses up to 10³ U/ml. rH-IFN-γ inhibited the survival of MKN-45 dose-dependently, while rH-IFN-α and -β did not inhibit the survival of MKN-45 even at the highest concentrations tested (10⁴ U/ml). Combination of rH-TNF with rH-IFN-α, -β or -γ did not significantly inhibit the survival of MKN-45, except for a combination of 10 U/ral of rH-TNF and 10³ U/ml of rH-IFN-γ ( P <0.05). Cisplatin inhibited the survival of MKN-45 dose-dependently. By the simultaneous combination of cisplatin with rH-TNF and/or rH-IFN-α, -β or γ, cytotoxicity of cisplatin was enhanced and the combination effects were additive. The effects of rH-TNF and rH-IFN-α, β and -γ on the modification of cytotoxicity of cisplatin were evaluated in terms of modification index (MI), demonstrating that rH-TNF, rH-IFN-α, -β and -γ all augmented the cytotoxicity of cisplatin: MI values at 10³ U/ml of rH-IFN-α, -β and -γ were 1.4, 1.4 and 2.3, respectively; those at the same concentrations of rH-IFN-α, -β and -γ in the presence of 10 U/ml of rH-TNF were 3.6, 2.5 and 5.1, respectively. These results demonstrating that the cytotoxicity of cisplatin was enhanced by rH-TNF and/or rH-IFN-α, -β or -γ suggest that cancer may be more effectively treated with the combination of cisplatin with these biological response modifiers than with cisplatin alone.     

Endogenous Tumor Necrosis Factor Induction with Bordetella pertussis Vaccine as a Triggering Agent and Its Therapeutic Effect on MM46 Carcinoma-bearing Mice

Induction of endogenous tumor necrosis factor (TNF) by administration of Bordetella pertussis vaccine (BPV) as a triggering agent and its therapeutic effect against MM46 carcinoma were investigated in C3H/He mice. Test triggering agents were injected intravenously into mice after intravenous injection of 4-fold dilution of macrophage activating factor (MAF) or 10⁴ units of murine interferon-γ (Mu-IFN-γ). Then sera were obtained from the mice, and their TNF activities were assayed on L-929 cells by the method of Ruff and Gifford. The triggering activity of BPV was the highest among those of conventional triggers, such as lipopolysaccharide (LPS) of Escherichia coli , and OK-432. The levels of serum TNF activity triggered by BPV (4 × 10⁹ cells), LPS of E. coli (3 μg) and OK-432 (3 KE) were 5350, 85 and 102 units/ml, respectively. Growth of MM46, a spontaneous mammary carcinoma cell line of C3H/He was observed for 35 days after tumor inoculation and was suppressed significantly by intravenous injection of MAF and BPV (4 × 10⁹ cells). On local injection of BPV (2 × 10⁹ cells) into murine tumors, complete regression was observed in 67% of the mice tested with or without MAF priming on day 25 after tumor inoculation, and intratumoral TNF activity was observed even in the case of the single injection of BPV.     

Antitumor Effect of PSK at a Distant Site: Tumor-specific Immunity and Combination with Other Chemotherapeutic Agents

The antitumor effect of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received intradermal inoculations of syngeneic Meth-A fibrosarcoma in the right (primary tumor, 10⁵ cells) and left (distant tumor, 2 × 10⁵ cells) flanks. Intratumoral administration of PSK significantly inhibited the growth of not only the right but also the left tumor. PSK also inhibited the growth of a methylcholanthrene-induced fibrosarcoma BAMC-1, and a methylurethane-induced adenocarcinoma Colon 26 in the double grafted tumor system of syngeneic BALB/c mice. However, when the left distant tumor was different from the right Meth-A tumor, the intratumoral administration of PSK in the right tumor was unable to inhibit the growth of the left' BAMC-1 or RL♂ -1 tumor. The PSK-induced immunity, therefore, is tumor-specific and T lymphocytes may play an important role in antitumor memory function. The enhancement of concomitant immunity by PSK treatment was completely impaired by previous intravenous administration of an alkylating agent, cyclophosphamide (CY). The enhancement of sinecomitant immunity by PSK treatment was also impaired by previous CY intravenous administration. The antitumor effect of PSK was suppressed by previous intravenous administration of another alkylating agent, ACNU. It is possible that alkylating agents suppress the function of effector T cells and granulocytes which are very important for the antitumor immune cascade reaction due to PSK treatment. On the other hand, the antitumor effect of PSK was enhanced by previous intravenous administration of an anti-metabolite, 5-fluorouraeil.     

Augmentation of Cancer Chemotherapy by Preinjection of Human Macrophage Colony-stimulating Factor in L1210 Leukemic Cell-inoculated Mice

Human macrophage colony-stimulating factor (hM-CSF) is a potent stimulator of the effector functions of monocytes/macrophages. We investigated the antitumor effects of this factor in CDF₁ male mice inoculated with L1210 cells, a mouse B-cell leukemia line. Mice preinoculated with various numbers of L1210 cells on day 0 were given intravenous injections of vehicle (human serum albumin; HSA) (100μg/kg/day) or hM-CSF (20μg/kg/day) for 3 days from day 1. In mice preinoculated with 10² L1210 cells but not with 10³ or more L1210 cells, a marked increment in survival rate was observed with hM-CSF treatment. We next examined the effect of hM-CSF treatment combined with chemotherapy on the survival of mice that had been preinoculated with 10⁵ L1210 cells. In our system, the administration of 4.9 mg/kg adriamycin (ADM) alone slightly prolonged survival of the tumorbearing mice, but all of the mice died within 20 days. When hM-CSF was injected for 3 days before this ADM treatment, the invasion and proliferation of tumor cells in the liver and spleen were markedly inhibited and 50% of the mice were still alive at day 50. We detected inhibitory activity toward L1210 growth in serum of mice administered with hM-CSF, and the degree of the inhibitory activity was correlated with the level of nitrite (NO₂) in the serum. When L1210 cells were co-cultured with peritoneal macrophages from mice intraperitoneally injected with hM-CSF, the uptake of [³H]thymidine in L1210 cells was inhibited. The inhibition was abolished by the addition of N^G-monomethyI-L-arginine, an inhibitor of NO₂− synthesis, suggesting that the reactive nitrogen oxide intermediate is involved in hM-CSF-induced inhibition of L1210 growth.     

Epidermal Growth Factor Receptor-mediated Selective Cytotoxicity of Antitumor Agents toward Human Xenografts and Murine Syngeneic Solid Tumors

Severe toxic side effects of antiproliferative agents limit their clinical usefulness as anti tumor drugs. Recently we observed that the anti tumor efficacy of various anti tumor agents (5-fluorouracil, tegafur, adriamycin, mitomycin C, cyclophosphamide, and cisplatin) against experimental solid tumors was enhanced by prior or simultaneous administration of human epidermal growth factor (EGF). However, coadministration of EGF did not enhance the toxicity of anti tumor agents as measured by LD₅₀ and body weight loss. The above selective potentiation of efficacy of the anti tumor agents by human EGF can be characterized as follows. In a dose-dependent manner, human EGF enhanced the efficacy of an antitumor agent (5-FU) treatment against human epidermoid carcinoma A431 transplanted sc in atliymic nude mice [ED₅₀=2.9 (0.2–49.7, 95% confidence interval) μg/kg, sc]. Various degrees of enhancement were also observed against other experimental tumors transplanted sc. The degrees of enhancement were directly proportional to the numbers of human EGF binding sites present on tumor cell plasma membrane (threshold of binding site density=1.5×10³ sites/cell) using 5-FU or cisplatin as an anti tumor agent, thus suggesting that the binding of EGF to the receptors on tumor cells is an essential process in enhancing the susceptibility of tumor cells to anti tumor agents. Normal cells including intestinal epithelial and bone marrow cells are endowed with fewer EGF binding sites (less than 10³ sites/cell). This may explain partially the absence of EGF-enhanced cytotoxicity by antitumor agents toward normal cells.     

Antitumor Effect of Interleukin-1β in the Double Grafted Tumor System

The antimetastatic effect of recombinant human interleukin-1β (rIL-1β) in a new experimental mouse model was studied. Intratumoral administration of IL-1β strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumor. Subsequently, the anti-metastatic effect of IL-1β was examined in the double grafted tumor system, in which mice first received simultaneous intradermal inoculations of Meth-A in both right (10⁶ cells) and left (2×10⁵ cells) flanks and were then injected with 0.2 μg of IL-1β in the right tumor on days 3, 4 and 5. IL-1β significantly inhibited the growth of the left, non-treated tumor. When mice received only an inoculation of Meth-A (2×10⁵ cells) in the left flank and were injected subcutaneously with IL-1β into the right flank on day 3 (single tumor system), there was no inhibition of the growth of the left, non-treated tumor. These findings suggest that intratumoral IL-1β immunotherapy in one region has an effect on tumor growth in another region. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of IL-1β. Adoptive transfer of the immunized spleen cells caused the complete regression of Meth-A tumors. These results suggest that intratumoral administration of IL-1β might induce cytotoxic cells in the left non-treated tumor of the double grafted tumor system and bring about the regression of metastatic tumors. On the other hand, recombinant tumor necrosis factor was effective only on the treated, right tumor, having no effect on the distant, left tumor in the double grafted tumor system. Recombinant interleukin-2 was effective on neither the right tumor nor the left tumor in this system. These results show that there are major differences of antitumor mechanism among cytokines.     

Augmentation of Murine Lymphokine-activated Killer Cell Induction by a Factor Produced by Nocardia rubra Cell Wall Skeleton-stimulated T Cells

Four-hour exposure of C3H/HeN mouse spleen cells to Nocardia rubra cell wall skeleton (N-CWS) before 4-day culture with a suboptimal dose of human recombinant interleukin 2 (rIL 2) augmented the induction of lymphokine-activated killer (LAK) cell activity, whereas the treatment with N-CWS alone induced no cytotoxicity. In accordance with this, the IL 2 binding activity of spleen cells was augmented by combined stimulation with N-CWS and rIL 2. The augmented cytotoxicity was mediated by Thy-1.2⁺, Lyt-1.1⁻, Lyt-2.1⁻ and asialo GM₁⁺ cells. Cell cultures in diffusion chambers revealed that N-CWS-treated spleen cells produced a LAK cell induction-helper factor (LAK-helper factor, LHF) when cultured with rIL 2. The LHF production required Thy-1.2⁺, Lyt-1.1⁺, Lyt-2.1⁺ and asialo GM₁⁻ cells, and the coexistence of unstimulated accessory cells was also essential for the LHF production. Cells responding to both LHF and rIL 2 to generate LAK activity were Thy-1.2⁻, Lyt-1.1⁻, Lyt-2.1⁻ and asialo GM₁⁺. The culture fluid of spleen cells stimulated with both N-CWS and rIL 2 contained no tumor necrosis factor (TNF) activity, and the additional stimulation with N-CWS caused no production of either IL 2 or interferon (IFN). Murine recombinant interleukin la (Mu-rIL 1α) could not replace the augmentative effect of N-CWS on LAK cell induction. These results suggest that in the presence of rIL 2, N-CWS stimulates murine T cells to produce LHF that is probably distinct from IL 1, IL 2, TNF and IFN.     

Recombinant Human Interferon-α2a Increases Hormone Receptor Level of a Human Breast Carcinoma Xenograft in Nude Mice and Enhances the Anti-proliferative Activity of Tamoxifen

The effect of recombinant human interferon-α2a (rhIFN-α2a) on the hormone receptor level and antitumor activity of tamoxifen (TAM) was investigated in nude mice using ZR-75-1, an estrogen receptor (ER)-positive, and progesterone receptor (PgR)-negative human breast carcinoma xenograft. ER levels (maximum binding sites) of tumors treated with rhIFN-α2a at a dose of 6 × 10⁴ U/mouse/ day for 1 or 3 wk were not significantly different from the control, whereas those with rhIFN-α2a at a dose of 6 × 10⁴ U/mouse/day for 1 or 3 wk were higher than the control (3.9- to 4.4-fold) with a significant difference at P < 0.01. The increase of ER by rhIFN-α2a was investigated using a sucrose density gradient method. The peak was only seen at 8S in both rhIFN-α2a-treated tumor and control ER, and the sedimentation patterns were almost the same, suggesting that both ERs were essentially equivalent. On the other hand, PgR of all the treated groups could be detected, while that of the control group was undetectable. The antitumor effect of the combination treatment of rhIFN-α2a and TAM was compared with those of single treatments. While rhIFN-α2a at a dose of 6 × 10⁵ U/mouse/day and TAM did not show a combination effect, rhIFN-α2a at a dose of 6 × 10⁴ U/mouse/day and TAM showed a synergistic combination effect, and ER was decreased to the threshold of detection by the combination treatment. These findings indicated that a low dose of rhIFN-α2a increased the ER levels of ER-positive human breast cancer in vivo as well as in vitro and enhanced the anti-proliferative effect of TAM, and the newly synthesized ER was essentially the same as the original ER.     

Subcutaneous Administration of Recombinant Human Granulocyte Colony-stimulating Factor (KRN8601) in Intensive Chemotherapy for Patients with Advanced Lung Cancer

The efficacy and toxicity of recombinant human granulocyte colony-stimulating factor (rh G-CSF, KRN8601) given subcutaneously was evaluated in patients with advanced lung cancer undergoing intensive chemotherapy. Twenty-nine and 30 patients with or without prior therapy were enrolled in this study. At dose levels of 50, 90 and 130 μg/m² of rh G-CSF for 14 consecutive days after chemotherapy, the mean neutrophil nadir counts, the mean neutrophil nadir ratios and the duration of neutropenia (days of < 1000/mm³) were significantly improved. No significant differences were seen in frequency and duration of febrile episodes (>38°C). When rh G-CSF is given subcutaneously, the dose required for an equal effect in alleviating neutropenia is 50% of that required when it is given intravenously. The monocyte counts in the peripheral blood were also significantly increased after chemotherapy cycles with rh G-CSF. The cumulative plasma concentration of rh G-CSF showed a decrement after 7–9 days despite maintenance of the same dose of rh G-CSF for the entire 14 days. In conclusion, 50–130 μg/m² of sc rh G-CSF increased the neutrophil nadir count and shortened the duration of neutropenia in patients undergoing intensive chemotherapy for lung cancer without intolerable side effects.     

Antitumor Effector Mechanism at a Distant Site in the Double Grafted Tumor System of PSK, a Protein-bound Polysaccharide Preparation

The antitumor effect at a distant site of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10⁶ cells) and left (2 × 10⁵ cells) flanks and were then injected with PSK in the right-flank tumor on day 3. PSK inhibited the growth of not only the right but also the left (non-treated) tumor. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of 5 mg of PSK and were injected into the Meth-A tumor on day 3. Adoptive transfer of PSK immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 monoclonal antibody plus complement. Spleen cells and right and left regional lymph node cells prepared from PSK immunized mice were examined for Thy-1, Lyt-1, Lyt-2 and asialo GM1 phenotypes. The number of Lyt-1-positive lymphocytes increased in the right regional lymph nodes after intratumoral administration of PSK. A massive accumulation of macrophages and polymorphonuclear leukocytes was found in the right tumor and an infiltration of macrophages and Lyt-2-positive lymphocytes was found in the left (non-treated) tumor by immunohistochemical analyses. These results suggest that intratumoral administration of PSK induces Lyt-1-positive cells first in regional lymph nodes, then in the spleen, and subsequently induces macrophages and Lyt-2-positive cells in the left (non-treated) tumor, thus bringing about the regression of metastatic tumors.     

Changes in Lymphocyte Subsets Following Multiple Administration of Recombinant Interleukin-2 plus Recombinant Interferon-beta or -gamma in Tumor-bearing Mice

Treatment with a combination of recombinant human interleukin-2 (rHIL-2) and recombinant mouse interferon-beta (rIFN-β) or -gamma (rIFN-γ) showed a significant antitumor effect against sc adenocarcinoma 755 in mice, although treatment with either one alone had almost no effect. The combination of rHIL-2 and rIFN-β caused regression of the tumor but the combination of rHIL-2 and rIFN-γ did not. Injection of tumor-bearing mice with the combinations of rHIL-2 and rIFN resulted in marked increases in the total number of peritoneal lymphocytes, and the frequency of Lyt-2⁺ cells was more markedly increased by the combination of rHIL-2 and rIFN-β than by the combination of rHIL-2 and rIFN-γ. In Winn assay, elimination of the Lyt-2⁺ population abolished the protective capacity of the peritoneal cells. The subsets of thymocytes were drastically changed when mice were bearing a tumor or were treated with cytokines. In particular, Lyt-2⁺/L3T4⁺ cells were decreased in tumor-bearing mice, but many Lyt-2⁺/L3T4⁺ cells were maintained in the thymus by treatment with a cytokine alone. When treated with rHIL-2 and rIFN-β the Lyt-2⁺/L3T4⁺ cells were markedly decreased, while Lyt-2^-/L3T4^- T-cells were increased, but these subsets were little changed by treatment with rHIL-2 plus rIFN-γ. Thus, injections of rHIL-2 and rIFN-β into tumor-bearing mice resulted in a high frequency of Lyt-2⁺/L3T4^- cells in the peritoneal cavity, together with changes in the T-cell subsets in the thymus. These results suggest that maturation of T-cells in the thymus may be an important step in the pathway by which cytokine treatment brings about regression of tumors.     

In vivo Tumor Growth Enhancement by Granulocyte Colony-stimulating Factor

The intraperitoneal administration of human recombinant granulocyte colony-stimulating factor (G-CSF) enhanced the growth of intradermally inoculated tumor in mice; in a Meth A fibrosarcoma model, G-CSF administration significantly shortened the latency before tumor appearance, accelerated the increase of tumor size, shortened the survival time of tumor-bearing mice and increased the incidence of lethal tumor growth. A similar growth-enhancing effect of G-CSF was observed in models employing Meth 1 fibrosarcoma, colon carcinoma 26, and L1210 leukemia, although not all the effects were statistically significant. In vitro study showed that G-CSF did not enhance Meth A growth in suspension culture or in soft agar. These data suggest that G-CSF enhances the Meth A growth not directly but through the mediation of host factors. The accumulation of neutrophils was histologically observed in the tumor nodule, the blood, and the spleen in mice given G-CSF repeatedly. The spleen cells and the peripheral blood leukocytes of G-CSF-injected mice enhanced Meth A growth in vitro as compared with those of mice injected with physiological saline. These results suggest the possibility that the in vivo growth of tumor cells was enhanced by G-CSF-induced overproduction of cells including neutrophils.     

Effect of Human Macrophage Colony-stimulating Factor on Granulopoiesis and Survival in Bone-marrow-transplanted Mice

Human macrophage colony-stimulating factor (hM-CSF) has been isolated from normal human urine and purified to a homogenous protein. The effect of hM-CSF on granulopoiesis was investigated in BALB/c mice transplanted with a suboptimal number of bone marrow cells. Lethally irradiated (7.8 Gy) mice were transplanted with 1 × 10⁶ syngeneic mouse bone marrow cells and treated with a daily intraperitoneal dose of 64 μg/kg of hM-CSF for 5 days following the transplant. The hM-CSF injection resulted in stimulation of the recovery of blood neutrophils as well as an increase in the number of granulocyte-macrophage progenitor cells (CFU-GM) in the femur and spleen. The survival of lethally irradiated mice was dependent on the cell number transplanted; most mice transplanted with 2 × 10⁴ cells died within 2 weeks. The recovery of hematopoiesis in mice transplanted with 2 × 10⁴ cells was modestly but significantly stimulated by hM-CSF administration initiated from 5 days before or 1 day after transplantation for a 5-day period. Furthermore, the hM-CSF administrations markedly reduced the mortality in these mice during the early period after the transplantation. Since anaerobic bacteria were frequently detected in arterial blood immediately before the deaths but were not found in the surviving mice, it is speculated that early deaths occurring within 2 weeks after the transplant may be caused by opportunistic infections, and hM-CSF injection may prevent these mortal infections through its stimulating effect on monocyte-macrophage functions that are responsible for the production of hematopoietic regulators.     

Induction Mechanism of Human Blood CD8+ T Cell Proliferation and Cytotoxicity by Natural Killer Cell Stimulatory Factor (Interleukin-12)

Natural killer cell stimulatory factor (NKSF J IL-12) has been found to induce cytotoxic activity of human blood T cells. In the present study, the effect of NKSF on induction of cytotoxic CD8⁺ T cells in the presence or absence of monocytes was examined. Highly purified lymphocytes (>99%) and monocytes (>90%) were isolated hy centrifugal elutriation from peripheral blood of normal donors. Then, CD8⁺ cells were isolated with antibody-bound magnetic beads from purified lymphocytes. The cytotoxicity of CD8⁺ cells was measured by ⁵¹Cr release assay for 4 h. NKSF enhanced the proliferative response of CD8⁺ cells stimulated with suboptimal concentrations of interleukin-2 (IL-2), but rather inhibited their proliferative and cytotoxic responses on stimulation with an optimal concentration of IL-2. NKSF stimulated CD8⁺ cells to produce interferon 7 (IFNγ) irrespective of the presence of added IL-2, and this effect was augmented by co-cultivation with monocytes. Blood monocytes upregulated induction of cytotoxic CD8⁺ cells stimulated with NKSF alone, and this effect was abolished by addition of antibody against IFNγ, but not of antibody against tumor necrosis factor a. Induction of NKSF-inducible cytotoxic CD8⁺ cells was inhibited by addition of transforming growth factor β, but not of IL-4. These observations suggest that in situ induction of NKSF-stimulated cytotoxic CD8⁺ cells may be regulated by complex cytokine networks, depending on the participation of monocytes.     

Antitumor Effect of Intratumoral Administration of Biological Response Modifiers: Induction of Immunosuppressive Acidic Protein, a Type of α1-Acid Glycoprotein, in Mice

The antitumor effects of biological response modifiers (BRMs) in an experimental mouse model, the "double grafted tumor system" were analyzed. Male BALB/c mice received simultaneous inoculations of Metn-A fibrosarcoma cells on the right flank (10⁶ cells) and left flank (2 × 10⁵ cells) on day 0, and BRMs were injected intratumorally into the right tumor on days 3, 4 and 5. PSK (a protein-bound polysaccharide preparation), interleuldn-1 (IL-1) and cepharanthin (R) cured not only the right, but also the left, non-treated tumor in a double grafted tumor system. OK-432 (a Streptococcus preparation) and BCG and tumor necrosis factor (TNF) cured the right tumor and inhibited the growth of the left tumor. Lentinan (a polysaccharide preparation) and IL-6 inhibited neither the right nor the left tumor. Immunosuppressive acidic protein (IAP) in serum was increased transiently soon after intradermal injection of PSK, CR, OK-432 and TNF in BALB/c mice. Lentinan, however, did not induce IAP. IAP in serum was gradually increased after intradermal inoculation of Meth-A tumor in BALB/c mice. The biochemical difference between PSK-induced IAP (early, inflammatory IAP) and Meth-A-induced IAP (late, tumor-induced IAP) was investigated by crossed affinity immunoelectrophoresis with concanavalin A. IAP of murine serum was separated into 4 peaks. IAP in normal mouse was rich in high-mannose type sugar chain (Peak 3) and contained no hybrid-type sugar chain (Peak 4), which was present in inflammatory and tumor-induced IAP. Inflammatory IAP was rich in biantennary sugar chain (Peak 2) and tumor-induced IAP was rich in tri-tetraantennary sugar chain (Peak 1).     

Granulocyte-colony Stimulating Factor and Retinoic Acid Cooperatively Induce Granulocytic Differentiation of Acute Promyelocytic Leukemia Cells in vitro

The interaction of granulocyte-colony stimulating factor (G-CSF) and retinoic acid (RA) in proliferation and differentiation of acute promyelocytic leukemia (APL) cells was examined. G-CSF stimulated proliferation of APL cells at concentrations of 0.1 to 50 ng/ml in a dose dependent manner. More than 10⁻⁸ M RA induced granulocytic differentiation of APL cells. Although G-CSF induced lysozyme activities in APL cells, it alone did not induce terminal differentiation of APL cells. G-CSF significantly enhanced the RA-induced granulocytic differentiation of APL cells in vitro. Enhancement by G-CSF was not due to the prolongation of survival of RA-induced differentiated cells, but the differentiation-inducing effects of G-CSF might be evident only in the presence of RA. Since G-CSF has a potential to induce the granulocytic differentiation of myeloid leukemia cells, G-CSF in combination with RA may be applicable in differentiation induction therapy for some types of myeloid leukemia.     

Enhancing effect by anti-cancer drugs on growth inhibition of colon carcinoma in nude mice by monoclonal antibody and complement

We investigated the possible sero-therapeutic application of monoclonal antibody-A7 against human colorectal cancer. In complement dependent cytotoxicity, A7 showed 59% cytotoxicity against SW1116 cells. In addition, the killing of tumor cells by A7 and C was enhanced when the tumor cells were pretreated with 2 micrograms/ml mitomycin and 40 micrograms/ml adriamycin. Next, we evaluated the in vivo antitumor effect of A7 alone and combined with MMC on human colon cancer (Colon-6) bearing nude mice. The group injected with A7 alone showed definite antitumor effect compared with the non-treated group. The A7+MMC group (MMC: 4mg/kg, A7: 1 mg/body, two times) showed enhanced antitumor effect compared with the groups administered A7 alone or MMC alone.